1. Field of the Invention
The technology disclosed herein relates to a sensor for detecting chemical substances or biological molecules in a specimen and, more specifically, to a biochemical sensor and a measuring device capable of detecting chemical substances or biological molecules in a specimen quickly in a simple manner.
2. Description of the Background Art
Recently, increase in lifestyle-related diseases has come to be a major problem. Health problems caused by environmental destruction are also increasing. Further, increasingly complex living conditions leads to larger population suffering from repression and psychic stress. In such a modern society, physical and mental health is naturally a matter of serious concern.
To maintain physical and mental health and to enjoy improved quality of life, symptomatic treatment, that is, treatment taken after one became aware of distinct abnormality, would be insufficient. To stay fit, prevention, such as taking a regular medical check-up, is desirable for early detection and early treatment of potential physical and mental abnormality.
Medical check-up involves collection, analysis and diagnosis of specimens. Conventionally, appropriate facility and well-trained personnel were considered indispensable to appropriately conduct these operations. As a result, it was a common practice to have a medical check-up at a hospital. Technical development, however, has enabled medical check-up in a manner different from the conventional method. Specifically, advancement in biotechnology and improvement in communication technology such as the Internet contribute to such new approach.
For instance, the following approach is now available. Specifically, the subject collects and analyzes a specimen at his/her home using a device that can be operated with little practice, and obtains data of the specimen. The subject transmits the data to a hospital through the Internet, and a doctor makes a diagnosis based on the transmitted data. The result of diagnosis is again sent to the subject at home through the Internet. Such medical check-up does not require a visit to the hospital, and hence, it is not time-consuming. Therefore, such an approach is convenient and effective to maintain one's physical and mental health.
In order to spread such method of medical check-up, however, it is necessary to establish technique necessary therefor. To enable medical check up as part of daily life, it is essential that the specimen can be collected and analyzed quickly in a simple manner. Therefore, a device for collecting the specimen for medical check up must be small and allow easy operation. Further, analysis of the specimen must also be done without requiring large-scale equipment.
At present, biochemical sensing techniques are promising to meet such demands. An example is a lavatory pan allowing checking of one's health condition based on excrements. Specific examples of other biochemical devices will be described in the following, considering physical aspects and mental aspects separately.
As regards the physical aspect, it is noted that the number of patients afflicted with lifestyle-related diseases has been increasing, due to poor diet and un-orderly lifestyle. A representative example is diabetes. For early detection and treatment of diabetes, and for improving quality of life of diabetic patients, it is essential to ascertain condition of the disease on a daily basis. To meet such a demand, a disposable glucose sensor that quickly detects blood glucose level useful for determining diabetic condition has been practically used. The glucose sensor is an example of the biochemical device.
As regards the mental aspect, more and more people are stressed-out in an increasingly complex society. Heavy stress may lead to grave consequences. To relieve stress as early as possible, first of all, it is necessary to know that one is under stress. For this purpose, a device that can accurately measure the stressed state of a user is desired. A specific method of detecting stress includes collection of blood, urine or saliva and detection of cortisol (one of vital hormones) as stress-related substance contained therein. A biochemical device is also used for detecting cortisol.
As described above, currently, there is a need for a technique that enables quick and simple diagnosis of both physical and mental health condition, and development of more advanced technique is desired. Therefore, development of biochemical technique that can detect information related to physical and mental health condition with high sensitivity, at high speed, in a simple manner and at a low cost is an important task. Such technique would be effective not only for medical check up but also for other applications.
As a technique realizing biochemical sensing with high sensitivity, use of antigen-antibody reaction and porous materials has been known. In one such technique, for the antigen as the target material to be detected (hereinafter simply referred to as the “target”), the antibody is carried adsorbed in the pores of the porous material. Application of such a technique is not limited to the antigen-antibody reaction, and it is generally applicable to two substances that react uniquely to each other. In the following description, however, examples will be described in which the antigen is the target and the antibody is the substance that is adsorbed and carried in the pores.
Porous silica is known as a material that realizes a structure for adsorbing and carrying the antibody and does not deactivate the antibody. Japanese Patent Laying-Open No. 2004-83501 discloses a bio-sensing technique using porous silica. Porous silica is formed by sol-gel method using organo-silicate as a main raw material or by freeze drying using inorgano-silicate. The former will be referred to as sol-gel porous silica, and the latter will be referred to as freeze-dried porous silica.
Such porous silica adsorbs the antibody in the minute pores of the porous material without damaging its function. Further, the porous silica is translucent. A biochemical sensor may be fabricated using the porous silica having such characteristics. In the following, a method of detecting an antigen using the biochemical sensor will be described.
First, a prescribed amount of antigen, fluorescence-labeled with fluorescent dye molecules and the like, is prepared as a standard sample. Next, sample specimen containing antigen as the target, which is not fluorescence-labeled, and the standard sample containing the antigen fluorescence-labeled with fluorescent dye molecules as described above are mixed. The mixed sample is applied to the porous silica having the antibody adsorbed and carried therein, to cause a reaction between the antigen in the mixed sample and the antibody adsorbed in the porous silica. By the antigen-antibody reaction, the antigen contained in the sample and the antibody adsorbed in porous silica are bonded. When the bonded antigen and antibody are irradiated with excitation light that excites the fluorescent dye molecules used for the fluorescence labeling, the antigen derived from the standard sample emits fluorescent light. By detecting intensity of the emitted fluorescent light using a photodetector, the amount of antigen contained in the sample specimen can be measured in the following manner.
The antigen in the mixed sample can be divided to two classes: those derived from the standard sample (fluorescence-labeled) and those derived from the sample specimen (not fluorescence-labeled). The amount of antigen derived from the standard sample is invariable. On the contrary, the amount of antigen derived from the sample specimen varies dependent on the amount of antigen in the sample specimen. Therefore, abundance ratio of the antigen derived from the standard sample and the antigen from the sample specimen varies. The amount of antibody in the porous silica is invariable. Accordingly, the abundance ratio of antigen from the standard sample and the antigen from the sample specimen of the whole antigen bonded to the antibody varies in accordance with the abundance ratio of antigen from the standard sample and the antigen from the sample specimen in the mixed sample. The fluorescent light is emitted only by the antigen derived from the standard sample, and therefore, the fluorescence intensity varies in accordance with the abundance ratio. As a result, the amount of antigen in the original sample specimen can be known from the fluorescence intensity, in the following manner.
A plurality of mixed samples having various different compositions of antigen fluorescence-labeled with fluorescent dye molecules and antigen not labeled are prepared beforehand. Fluorescent light detection is done in the manner as described above for each of these mixed samples. Based on the thus obtained fluorescence intensities, a calibration curve is formed. The calibration curve indicates, as a graph, expected fluorescence intensity based on the mixture ratio of the labeled antigen and unlabeled antigen. When there is an unknown amount of antigen fluorescence-labeled with fluorescent dye molecules, it is possible to know the amount of antigen, by mixing the antigen with a known amount of standard sample, measuring variation in the fluorescence intensity, and comparing the intensity with the calibration curve.
The prior art disclosed in Japanese Patent Laying-Open No. 2004-83501 mentioned above relates to a technique of fixing antibody in minute pores in the sol-gel porous silica having minute pores formed in the form of a film. By this technique, the antibody can stably be fixed.
Even in this method, however, the amount of antibody that can effectively be carried in the minute pores and utilized for measurement is limited by the area of the minute pores formed as a film on a surface of a base material. This limitation leads to insufficient accuracy of antigen detection.
Further, generally it takes time for the antigen to penetrate and reach the antibody adsorbed and carried in the minute pores and to be bonded to the antibody. As the rate of detection is controlled by the rate of penetration, it is difficult to realize highly sensitive and quick sensing by the technique described in Japanese Patent Laying-Open No. 2004-83501.
J. C. Zhou, et al., Immunoassays for cortisol using antibody-doped sol-gel silica, J. Mater. Chem., 2004, 14, 2311-2316 discloses a solution to the problems of insufficient detection sensitivity and difficulty in highly sensitive and quick sensing. The technique disclosed by Zhou et al. is directed to a porous silica structure adsorbing and carrying a substance functionalized to react to a prescribed substance such as the antibody and to generate a reactant.
The object of the invention disclosed by Zhou et al. is to detect cortisol. Zhou et al. further discloses a technique utilizing antibody carried by porous silica gel formed by sol-gel method using organo-silicate raw material.
According to Zhou et al., to fabricate the porous structure, first, tetramethylorthosilicate, water and diluted hydrochloric acid are mixed, to prepare silica hydrosol. The mixture is further mixed with phosphate buffer containing anti-cortisol antibody. By sol-gel method of gelating the mixture, a porous silica monolith body containing the anti-cortisol antibody is formed.
By the technique disclosed by Zhou et al., a porous silica monolith body is formed that adsorbs and carries anti-cortisol antibody in the minute pores. Therefore, as compared with the disclosure of Japanese Patent Laying-Open No. 2004-83501 in which the antibody is adsorbed and carried by the porous material formed as a film on a surface of a base material, sensitivity of antigen detection can be improved. The disclosure of Zhou et al., however, still has a limitation in the amount of antibody that can be used for measurement, and hence, detection sensitivity is insufficient. The reason will be described in the following.
When antigen molecules are applied to the monolith body, the antigen molecules may react rather quickly to the antibody molecules adsorbed and carried near the surface of the monolith body. For the antigen to reach and react to the antibody adsorbed and carried at positions deep inside from the surface, it is necessary that the antigen molecules pass through the porous mesh structure and to diffuse deep inside the monolith body. It naturally takes a long time for the antigen molecules to reach the deep position in the monolith body and, as a result, the antigen-antibody reaction also takes long time.
In order to solve this problem, it may be possible to fabricate a porous silica monolith body that is relatively thin (having the thickness, for example, of 1 mm). Such a monolith body, however, still has a problem. Specifically, the diffusion length in the monolith body is typically in proportion to the root of diffusion time. When compared with a monolith thin film formed of the same material and having the thickness of 10 μm, passage through the monolith body of 1 mm takes 104 times longer time. Specifically, when a sol-gel silica monolith body of a simple structure is used, the antigen cannot sufficiently penetrate to the antibody existing at a deep position until after a long time, even if the thickness is as thin as 1 mm. Therefore, the amount of antibody available for reaction with the antigen for a short period of time is limited to the antibody adsorbed and carried on and near the surface of the monolith body. In such a biochemical sensor, the amount of antibody that can react to the antigen is substantially in proportion to the surface area of the monolith body. As a result, if the monolith body has small surface area, the amount of antigen-antibody reaction becomes small, and hence, it is difficult to realize a sensor capable of highly sensitive detection. On the other hand, if the surface area is made larger, handling of the monolith body becomes troublesome.
Specifically, in the technique disclosed in Zhou et al., when antigen detection is to be done effectively utilizing antibody carried at positions deep inside the monolith body, the time for measurement becomes undesirably long. When the time for measurement is to be reduced while the monolith body has small surface area, detection intensity would be insufficient. When the surface area of the monolith body is increased, handling becomes troublesome.
Conditions related to sensor sensitivity will be described with reference to specific examples. As an example of detection of very small amount of target, consider detection of stress-related substance. An example of such substance is cortisol in saliva. In order to obtain significant data of cortisol (MW:362) in saliva, typically, measurement of concentration in the range of about 1.0 to about 30.0 pmol/ml is necessary. The technique disclosed in Zhou et al. allows detection of cortisol in saliva only in the range of 27.6 pmol to 2.76 nmol/ml. From the viewpoint of obtaining significant data, necessary measurable ranges of other substances are as follows. As to human salivary chromogranin A (human CgA) (MW:68000), detection of a small amount of about 0.4 to 1.2 pmol/ml is necessary. As to immuno globulin (MW:200000), detection of a small amount of about 0.5 to 5.0 nmol/ml is necessary.
The technique disclosed in Zhou et al. is advantageous in that, compared with the amount of antibody adsorbed and carried on the porous body formed only at and near the surface of the base material, larger amount of antibody can be adsorbed. It is still impossible, however, to detect the necessary amount of substance with sufficient sensitivity in a short period of time, and to attain higher sensitivity, detection requires long time.